1. Field of the Invention
This invention relates to novel multibinding compounds (agents) that are xcex22 adrenergic receptor agonists and pharmaceutical compositions comprising such compounds. Accordingly, the multibinding compounds and pharmaceutical compositions of this invention are useful in the treatment and prevention of respiratory diseases such as asthma and chronic bronchitis. They are also useful in the treatment of nervous system injury and premature labor.
2. References
The following publications are cited in this application as superscript numbers:
1Hardman, J. G. , et al. xe2x80x9cThe Pharmacological Basis of Therapeuticsxe2x80x9d, McGraw-Hill, New York, (1996)
2Strosberg, A. D. xe2x80x9cStructure, Function, and Regulation of Adrenergic Receptorsxe2x80x9d Protein Sci. 2, 1198-1209 (1993).
3Beck-Sickinger, A. G. xe2x80x9cStructure Characterization and Binding Sites of G-Protein-coupled Receptorsxe2x80x9d DDT, 1, 502-513, (1996).
4Hein, L. and Kobilka, B. K. xe2x80x9cAdrenergic Receptor Signal Transduction and Regulationxe2x80x9d Neuropharmacol, 34, 357-366, (1995).
5Strosberg, A. D. and Pietri-Rouxel, F. xe2x80x9cFunction, and Regulation of xcex23 Adrenoceptorxe2x80x9d TiPS, 17, 373-381, (1996).
6Barnes, P. J. xe2x80x9cCurrent Therapies for Asthmaxe2x80x9d CHEST, 111:17S-26S, (1997).
7Jack, D. A. xe2x80x9cA way of Looking at Agonism and Antagonism: Lessons from Salbutamol, Salmeterol and other xcex2-Adrenoceptor Agonistsxe2x80x9d Br. J Clin. Pharmac. 31, 501-514, (1991).
All of the above publications are herein incorporated by reference in their entirety to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference in its entirety.
3. State of the Art
A receptor is a biological structure with one or more binding domains that reversibly complexes with one or more ligands, where that complexation has biological consequences. Receptors can exist entirely outside the cell (extracellular receptors), within the cell membrane (but presenting sections of the receptor to the extracellular milieu and cytosol), or entirely within the cell (intracellular receptors). They may also function independently of a cell (e.g., clot formation). Receptors within the cell membrane allow a cell to communicate with the space outside of its boundaries (i.e., signaling) as well as to function in the transport of molecules and ions into and out of the cell.
A ligand is a binding partner for a specific receptor or family of receptors. A ligand may be the endogenous ligand for the receptor or alternatively may be a synthetic ligand for the receptor such as a drug, a drug candidate or a pharmacological tool.
The super family of seven transmembrane proteins (7-TMs), also called G-protein coupled receptors (GPCRs), represents one of the most significant classes of membrane bound receptors that communicate changes that occur outside of the cell""s boundaries to its interior, triggering a cellular response when appropriate. The G-proteins, when activated, affect a wide range of downstream effector systems both positively and negatively (e.g., ion channels, protein kinase cascades, transcription, transmigration of adhesion proteins, and the like).
Adrenergic receptors (AR) are members of the G-protein coupled receptors that are composed of a family of three receptor sub-types: xcex11 (A, B, D) xcex12 (A, B, C) and xcex2 (1, 2, 3).1-5 These receptors are expressed in tissues of various systems and organs of mammals and the proportions of the xcex1 and the xcex2 receptors are tissue dependant. For example, tissues of bronchial smooth muscle express largely xcex22-AR while those of cutaneous blood vessels contain exclusively xcex1-AR subtypes.
It has been established that the xcex22-AR sub-type is involved in respiratory diseases such as such as asthma6, chronic bronchitis, nervous system injury and premature labor. Currently, a number of drugs e.g., albuterol, formeterol, isoprenolol, or salmeterol having xcex22-AR agonist activities are being used to treat asthma. However, these drugs have limited utility as they are either non-selective thereby causing adverse side effects such as muscle tremor, tachycardia, palpitations, and restlesness6, or have short duration of action and/or slow onset time of action.7 Accordingly, there is a need for xcex22-selective AR agonists that are fast acting and have increased potency and/or longer duration of action.
The multibinding compounds of the present invention fulfill this need.
This invention is directed to novel multibinding compounds (agents) that are xcex22 adrenergic receptor agonists and are therefore useful in the treatment and prevention of respiratory diseases such as asthma and chronic bronchitis. They are also useful in the treatment of nervous system injury and premature labor.
Accordingly, in one of its composition aspects, this invention provides a multibinding compound of Formula (I):
xe2x80x83(L)p(X)qxe2x80x83xe2x80x83(I)
wherein:
p is an integer of from 2 to 10;
q is an integer of from 1 to 20;
L is a ligand wherein:
one of the ligands, L, is a compound of formula (a): 
herein:
Ar1 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar1 substituent optionally links the ligand to a linker;
R1 is selected from the group consisting of hydrogen, alkyl, and substituted alkyl, or R1 is a covalent bond linking the ligand to a linker;
R2 and R3 are independently selected from the group consisting of hydrogen, alkyl, acyl, and substituted alkyl, or R2 and/or R3 are a covalent bond linking the ligand to a linker; and
the other ligands are independently selected from a compound of formula (b): 
wherein:
Ar2 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar2 substituent optionally links the ligand to a linker;
R11 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, or R11 a covalent bond linking the ligand to a linker;
R12 and R13 are independently selected from the group consisting of hydrogen, alkyl, acyl, or substituted alkyl, or R12 and/or R13 are a covalent bond linking the ligand to a linker;
each linker, X, in the multibinding compound of Formula (I) independently a straight or branched alkylene chain provided that when the multibinding compound of Formula (I) is a compound of formula (c): 
where Ar1 and Ar2 are aryl then X is not:
(i) a straight alkylene chain of 2-12 carbon atoms or a branched alkylene chain of formula xe2x80x94C(RaRa)xe2x80x94(CH2)mxe2x80x94C(Ra)(Ra)xe2x80x94 where Ra is methyl and m is 0 or an integer of from 1-8;
and pharmaceutically acceptable salts thereof.
Preferably, q is less than p in the multibinding compounds of this invention.
In still another of its composition aspects, this invention provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a multibinding compound of Formula (I):
(L)p(X)qxe2x80x83xe2x80x83(I)
wherein:
p is an integer of from 2 to 10;
q is an integer of from 1 to 20;
L is a ligand wherein:
one of the ligands, L, is a compound of formula (a): 
wherein:
Ar1 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar1 substituent optionally links the ligand to a linker;
R1 is selected from the group consisting of hydrogen, alkyl, and substituted alkyl, or R1 is a covalent bond linking the ligand to a linker;
R2 and R3 are independently selected from the group consisting of hydrogen, alkyl, acyl, and substituted alkyl, or R2 and/or R3 are a covalent bond linking the ligand to a linker; and
the other ligands are independently selected from a compound of formula (b): 
wherein:
Ar2 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar2 substituent optionally links the ligand to a linker;
R11 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, or R11 a covalent bond linking the ligand to a linker;
R12 and R13 are independently selected from the group consisting of hydrogen, alkyl, acyl, or substituted alkyl, or R12 and/or R13 are a covalent bond linking the ligand to a linker;
each linker, X, in the multibinding compound of Formula (I) independently a straight or branched alkylene chain provided that when the multibinding compound of Formula (I) is a compound of formula (c): 
where Ar1 and Ar2 are aryl then X is not:
(i) a straight alkylene chain of 2-12 carbon atoms or a branched alkylene chain of formula xe2x80x94C(RaRa)xe2x80x94(CH2)mxe2x80x94C(Ra)(Ra)xe2x80x94 where Ra is methyl and m is 0 or an integer of from 1-8;
and pharmaceutically acceptable salts thereof.
In still another aspect, this invention provides a method of treating diseases mediated by a xcex22 adrenergic receptor in a mammal, said method comprising administering to said mammal a therapeutically effective amount of a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a multibinding compound of Formula (I):
(L)p(X)qxe2x80x83xe2x80x83(I)
wherein:
p is an integer of from 2 to 10;
q is an integer of from 1 to 20;
L is a ligand wherein:
one of the ligands, L, is a compound of formula (a): 
wherein:
Ar1 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar1 substituent optionally links the ligand to a linker;
R1 is selected from the group consisting of hydrogen, alkyl, and substituted alkyl, or R1 is a covalent bond linking the ligand to a linker;
R2 and R3 are independently selected from the group consisting of hydrogen, alkyl, acyl, and substituted alkyl, or R2 and/or R3 are a covalent bond linking the ligand to a linker; and
the other ligands are independently selected from a compound of formula (b): 
wherein:
Ar2 is selected from the group consisting of aryl, heteroaryl, cycloalkyl, substituted cycloalkyl, and heterocyclyl wherein each of said Ar2 substituent optionally links the ligand to a linker;
R11 is selected from the group consisting of hydrogen, alkyl, substituted alkyl, or R11 a covalent bond linking the ligand to a linker;
R12 and R13 are independently selected from the group consisting of hydrogen, alkyl, acyl, or substituted alkyl, or R12 and/or R13 are a covalent bond linking the ligand to a linker;
each linker, X, in the multibinding compound of Formula (I) independently a straight or branched alkylene chain provided that when the multibinding compound of Formula (I) is a compound of formula (c): 
where Ar1 and Ar2 are aryl then X is not:
(i) a straight alkylene chain of 2-12 carbon atoms or a branched alkylene chain of formula xe2x80x94C(RaRa)xe2x80x94(CH2)mxe2x80x94C(Ra)(Ra)xe2x80x94 where Ra is methyl and m is 0 or an integer of from 1-8;
and pharmaceutically acceptable salts thereof.
In still another aspect, this invention is directed to general synthetic methods for generating large libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties for xcex22 adrenergic receptor. The diverse multimeric compound libraries provided by this invention are synthesized by combining a linker or linkers with a ligand or ligands to provide for a library of multimeric compounds wherein the linker and ligand each have complementary functional groups permitting covalent linkage. The library of linkers is preferably selected to have diverse properties such as valency, linker length, linker geometry and rigidity, hydrophilicity or hydrophobicity, amphiphilicity, acidity, basicity and polarization. The library of ligands is preferably selected to have diverse attachment points on the same ligand, different functional groups at the same site of otherwise the same ligand, and the like.
This invention is also directed to libraries of diverse multimeric compounds which multimeric compounds are candidates for possessing multibinding properties for xcex22 adrenergic receptor. These libraries are prepared via the methods described above and permit the rapid and efficient evaluation of what molecular constraints impart multibinding properties to a ligand or a class of ligands targeting a receptor.
Accordingly, in one of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for xcex22 adrenergic receptor which method comprises:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties for xcex22 adrenergic receptor.
In another of its method aspects, this invention is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for xcex22 adrenergic receptor which method comprises:
(a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand;
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands; and
(d) assaying the multimeric ligand compounds produced in (c) above to identify multimeric ligand compounds possessing multibinding properties for xcex22 adrenergic receptor.
The preparation of the multimeric ligand compound library is achieved by either the sequential or concurrent combination of the two or more stoichiometric equivalents of the ligands identified in (a) with the linkers identified in (b). Sequential addition is preferred when a mixture of different ligands is employed to ensure heterodimeric or multimeric compounds are prepared. Concurrent addition of the ligands occurs when at least a portion of the multimer comounds prepared are homomultimeric compounds.
The assay protocols recited in (d) can be conducted on the multimeric ligand compound library produced in (c) above, or preferably, each member of the library is isolated by preparative liquid chromatography mass spectrometry (LCMS).
In one of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties for xcex22 adrenergic receptor which library is prepared by the method comprising:
(a) identifying a ligand or a mixture of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a library of linkers wherein each linker in said library comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the ligand or mixture of ligands identified in (a) with the library of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In another of its composition aspects, this invention is directed to a library of multimeric ligand compounds which may possess multivalent properties for xcex22 adrenergic receptor which library is prepared by the method comprising:
(a) identifying a library of ligands wherein each ligand contains at least one reactive functionality;
(b) identifying a linker or mixture of linkers wherein each linker comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand; and
(c) preparing a multimeric ligand compound library by combining at least two stoichiometric equivalents of the library of ligands identified in (a) with the linker or mixture of linkers identified in (b) under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands.
In a preferred embodiment, the library of linkers employed in either the methods or the library aspects of this invention is selected from the group comprising flexible linkers, rigid linkers, hydrophobic linkers, hydrophilic linkers, linkers of different geometry, acidic linkers, basic linkers, linkers of different polarization and amphiphilic linkers. For example, in one embodiment, each of the linkers in the linker library may comprise linkers of different chain length and/or having different complementary reactive groups. Such linker lengths can preferably range from about 2 to 100 xc3x85.
In another preferred embodiment, the ligand or mixture of ligands is selected to have reactive functionality at different sites on said ligands in order to provide for a range of orientations of said ligand on said multimeric ligand compounds. Such reactive functionality includes, by way of example, carboxylic acids, carboxylic acid halides, carboxyl esters, amines, halides, isocyanates, vinyl unsaturation, ketones, aldehydes, thiols, alcohols, anhydrides, and precursors thereof. It is understood, of course, that the reactive functionality on the ligand is selected to be complementary to at least one of the reactive groups on the linker so that a covalent linkage can be formed between the linker and the ligand.
In other embodiments, the multimeric ligand compound is homomeric (i.e., each of the ligands is the same, although it may be attached at different points) or heterodimeric (i.e., at least one of the ligands is different from the other ligands).
In addition to the combinatorial methods described herein, this invention provides for an iterative process for rationally evaluating what molecular constraints impart multibinding properties to a class of multimeric compounds or ligands targeting a receptor. Specifically, this method aspect is directed to a method for identifying multimeric ligand compounds possessing multibinding properties for xcex22 adrenergic receptor which method comprises:
(a) preparing a first collection or iteration of multimeric compounds which is prepared by contacting at least two stoichiometric equivalents of the ligand or mixture of ligands which target a receptor with a linker or mixture of linkers wherein said ligand or mixture of ligands comprises at least one reactive functionality and said linker or mixture of linkers comprises at least two functional groups having complementary reactivity to at least one of the reactive functional groups of the ligand wherein said contacting is conducted under conditions wherein the complementary functional groups react to form a covalent linkage between said linker and at least two of said ligands;
(b) assaying said first collection or iteration of multimeric compounds to assess which if any of said multimeric compounds possess multibinding properties for xcex22 adrenergic receptor;
(c) repeating the process of (a) and (b) above until at least one multimeric compound is found to possess multibinding properties for 132 adrenergic receptor;
(d) evaluating what molecular constraints imparted multibinding properties to the multimeric compound or compounds found in the first iteration recited in (a)-(c) above;
(e) creating a second collection or iteration of multimeric compounds which elaborates upon the particular molecular constraints imparting multibinding properties to the multimeric compound or compounds found in said first iteration;
(f) evaluating what molecular constraints imparted enhanced multibinding properties to the multimeric compound or compounds found in the second collection or iteration recited in (e) above;
(g) optionally repeating steps (e) and (f) to further elaborate upon said molecular constraints.
Preferably, steps (e) and (f) are repeated at least two times, more preferably at from 2-50 times, even more preferably from 3 to 50 times, and still more preferably at least 5-50 times.